Chromosome 8q as the most frequent target for amplification in early gastric carcinoma.

Early gastric carcinoma (GC) is considered to be a curable cancer, as it progresses to the advanced stage following varying durations. Understanding the early stage of GC may provide an insight into its pathogenesis and contribute to reducing the mortality rate of this disease. To investigate the genomic aberrations associated with 22 cases of early GC, high-density microarray comparative genomic hybridization was performed in the present study. The most notable finding was copy number gains (log2 ratio >0.25) on the long arm of chromosome 8, which occurred in 77.3% (17/22) of GC cases, and the delineated minimal common region was 8q22.1-q24.3. More specifically, two amplified (log2 ratio >1) loci in the 8q22.1-q24.3 region were detected in 18.2% (4/22) of GC cases. The first loci covered a region of 102.4-107.9 kb, mapping on 8q22.3-q23.1, and comprised the transcription factor CP2-like 3 gene. The second loci, spanning 128.7-145.7 kb on 8q24.21-q24.3, comprised the representative oncogene of myelocytomatosis. Furthermore, the following possible target genes that were not previously considered to play a pathogenic role in GC were identified: Plasmacytoma variant translocation 1, cysteine/histidine rich 1, kinesin family member C2, forkhead box H1, protein phosphatase 1 regulatory subunit 16A, glutamic-pyruvate transaminase, LOC113655 and RecQ protein-like 4. In the present study, previous findings showing that 8q mutations accumulate early during the multistage pathogenesis of GC were confirmed and expanded upon. The confirmation of previously reported 8q gains and the identification of novel target genes at 8q22.1-q24.3 amplified chromosomal sites should aid in improving our understanding of the molecular mechanisms underlying the tumorigenesis of early GC.

Characterization of amplification patterns and target genes on the short arm of chromosome 7 in early-stage lung adenocarcinoma.

Chromosomal alterations are a predominant genomic force contributing to the development of lung adenocarcinoma (ADC). High density genomic arrays were conducted to identify critical genetic landmarks that may be important mediators in the formation or progression of early­stage ADC. In this study, the most noteworthy and consistent observation was a copy number gain on the short arm of chromosome 7, which was detected in 85.7% (12/14) of cases. Notably, three distinct regions of amplification were identified between the 7p22.3 and q11.2 regions in 28.6% (4/14) of cases; at a size of 4.1 Mbp (7p22.3­p21.1), 2.6 Mbp (7p15.2-p14.1) and 1.5 Mbp (7p12.3­p11.2). Variations of the 7p11.2 locus that encodes EGFR are known to be oncogenic. Furthermore, potential target genes were identified that were previously not assumed to be involved in the pathogenesis of ADC, including CALM1P2 (7p11.2), HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11 and HOXA13 (7p15.2) and LOC442586, LOC442589, LOC442282, FAM20C and LOC442651 (7p22.3). The present study determined critical regions on the 7p arm of chromosome 7, which were implicated in ADC. The pattern of rearrangements on the 7p arm may be a consequence of the high density of potential targets and the identified genes at the 7p regions may aid in the development of therapeutic targets for ADC.

Clinical implementation of chromosomal microarray technology in prenatal diagnosis. (Review).

Chromosomal microarray technology represents the technical convergence of molecular genetics and cytogenetics, and is rapidly revolutionizing modern cytogenetics. Expected genomic aberrations are accurately identified and provide readily interpretable results that are suitable for clinical risk stratification and therapeutic strategies. The application of array technology in prenatal genetic diagnosis provides distinct advantages over conventional cytogenetic analysis in detecting both the majority of microscopic and submicroscopic chromosomal abnormalities. In the last few years, the validity of array technology has become obvious to medical and laboratory communities involved in prenatal diagnostic testing. However, whether or not microarray analysis is sufficient for the detection of cytogenetic abnormalities in prenatal diagnosis and if traditional cytogenetics continue to be important in this new era has yet to be confirmed. In the present study, we systematically reviewed the current status of microarray technology in the identification of pathogenic genomic imbalances and discussed practical considerations for its routine implementation in prenatal diagnosis.

Evolving applications of microarray technology in postnatal diagnosis (review).

Microarray-based cytogenetics is revealing the tremendous fluidity and complexity of the human genome, and is starting to illustrate the implications of genomic variability with respect to human health and disease. In the last few years, the robustness of array-based technologies has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner for identifying genomic defects of congenital and developmental abnormalities including developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASD) and/or multiple congenital anomalies (MCA). The implementation of this technology in these categories of disorders has been thoroughly evaluated and is now recommended as a first-line diagnostic approach for clinically suspected genetic disorders. However, clinical application of array-CGH in postnatal evaluation raises the debate of whether array-CGH will replace traditional cytogenetics in the near future and whether there is still a role for karyotyping and FISH. In this article, we therefore review the current status of array-based technology use for postnatal diagnosis and predict that it will replace standard cytogenetics as a first-line test for clinical evaluation in these population groups.

ORAOV1 is a probable target within the 11q13.3 amplicon in lymph node metastases from gastric adenocarcinoma.

The lymph node metastatic (LNM) spread of tumor cells is a frequent event in the initial process of cancer dissemination and is a powerful independent prognostic indicator in gastric adenocarcinoma (GAC). High density genomic arrays were conducted to identify molecular markers associated with lymph node metastasis in GAC. In the genome-wide profile, large copy number gains involving chromosomes 1p, 3q, 8q, 9q, 11q, 16p, 19p, and 20q (log2 ratio >0.25) (>40% of patients) were more prevalent than copy number losses. The most notable finding was copy number gains at the long arm of chromosome 11, which occurred in 75.0% of lymphatic metastasis GAC cases, and the delineated minimal common region was 11q24.2-q12.1. More specifically, 2 amplified (>1 log2 ratio) loci on the 11q13.3 region were detected in 12.5% of the cases. The first locus, covers a region of ~7.7 Mbp, and comprises the representative oncogene of cyclin D1 (CCNDI). This finding occurred in 12.5% of the cases. Additionally, an oral cancer overexpressed 1 (ORAOV1) gene was identified as a probable target within the 11q13 amplicon, which previously was not assumed to play a pathogenic role in GACs (12.5%). A second locus spanning 7.8 Mbp on 11q13.3 without associated genes also showed high-level amplifications in 12.5% of the GACs. This study indicates that the long arm of chromosome 11 harbors protooncogenes that are associated with lymphatic metastasis formation and the ORAOV1 gene at the 11q13.3 region could be a potential target and serve as an indicator for the presence of occult metastases in GAC.

Assessment of the beneficial loci and prognostic implications of microsatellite instability in gastric carcinoma.

Microsatellite instability (MSI) is a hyper-mutable phenotype caused by the loss of DNA mismatch repair activity, and plays a crucial role in gastric carcinogenesis. To clarify the role of genetic instability in relation to clinicopathological variables, and to identify predictive MSI markers that facilitate the early detection and improve the classification of gastric carcinomas (GCs), 13 microsatellite (MS) loci, including the National Cancer Institute (NCI) panel of MS markers (D2S123, D5S346, D17S250, BAT25 and BAT26) and 8 dinucleotide repeats (D3S1260, D5S107, D5S409, D17S261, D17S520, D17S855, D18S34 and D18S61) were studied in GC patients. MSI was found in 88.2% (30/34) of GC cases and the number of high-frequency MSI (MSI-H, 23.5%, 8/34), low-frequency MSI (64.7%, 22/34), and stable MSI (11.8%, 4/34), was calculated. Among the MS loci analyzed, D18S34 and D17S261 (15/34, 44.1%) exhibited the highest frequency of MSI, followed by D2S123 (14/34, 41.2%), D5S107, D5S346, D5S409 and D18S61 (12/34, 35.3%) (MSI>35%). MSI-H was particularly prevalent in older patients and was mainly found in the antrum and poorly differentiated tumors. Furthermore, MSI-H was significantly associated with lymph node involvement cases in females. One notable finding in this analysis was that the markers, D17S250 and D17S520, exhibited a significantly higher percentage of MSI in advanced gastric carcinomas than in early gastric carcinomas (P=0.046 and 0.046, respectively), and the D17S520 and BAT26 loci represented significant different correlations between the tumor stages (P=0.038 and 0.042, respectively). This study indicates that the novel markers, D18S34 and D17S261, perform more favorably than the NCI panel for the detection of MSI, and the D17S520 locus presents a potential target for predicting the clinical impact of GC. These novel MS loci may prove to be beneficial and independent tools for the construction of a comprehensive genetic classification for GC cases.

Frequent silence of chromosome 9p, homozygous DOCK8, DMRT1 and DMRT3 deletion at 9p24.3 in squamous cell carcinoma of the lung.

Chromosomal alterations are a major genomic force contributing to the development of lung cancer. We subjected 22 cases of squamous cell carcinoma of the lung (SCC) to whole-genome microarray-CGH (resolution, 1 Mb) to identify critical genetic landmarks that might be important mediators in the formation or progression of SCC. On a genome-wide profile, copy number losses (log2 ratio <-0.25) on chromosome 9p occurred in 72.7% (16/22) of the SCC cases, and the delineated minimal common region was 9p24.3-p21.1. The progression of SCC to advanced stages or poorly differentiated malignancy was significantly associated with an increase in the copy number losses on 9p (P=0.033). More specifically, 2 distinct homozygous deletion (HD) loci (log2 ratio <-1) were identified as novel loci for candidate tumor suppressor genes (TSGs) in SCC: one, spanning 128 kbp on 9p21.1 [4.5% (1/22)] and the other, spanning approximately 200 kbp on 9p24.3 [13.6% (3/22)]. The HDs at 9p24.3 was found to contain the putative TSG, DOCK8 and 2 possible candidate TSGs, DMRT1 and DMRT3. Quantitative real-time PCR (qRT-PCR) analysis demonstrated the array-CGH-detected potential candidate genes DMRT1, DMRT3 and DOCK8, at 9p24.3 were under-expressed in SCCs. Our study indicated that chromosome 9p loss is the hallmark of SCC, and DMRT1, DMRT3 and DOCK8 genes at 9p24.3 might be of interest for the study of the pathophysiology of SCC as potential targets for therapeutic measures.

AMY2A: a possible tumor-suppressor gene of 1p21.1 loss in gastric carcinoma.

Homozygous deletions (HDs) are major genomic forces contributing to the development of many solid tumors. To identify critical tumor-suppressor loci involved in the pathogenesis of gastric carcinoma (GC), a high-resolution microarray-CGH was performed in a series of 27 GC patients. On a genome-wide profile, five distinct HD (log2 ratio <-1) loci, including 1p21.1, 2q21.1, 10q24.32, 13q34 and 15q11.2 were identified. These regions contained representative tumor-related genes, such as the FGF8 and NPM3 genes at 10q24.32, and the LAMP1 gene at 13q34, which have been reported in connection with various tumors. The most frequent HD encompassed chromosome band 1p21.1 in 5 of 27 GC cases (18.5%). A hemizygous deletion (-0.5< log2 ratio < or = -1) or a single copy loss (log2 ratio <-0.25) from the 1p21.1 region was noted in 51.9% (14/27) and 88.9% (24/27) of GCs, respectively. A 30 Kb HD of the 1p21.1 chromosomal region was shown to contain a potential candidate tumor-suppressor gene (TSG) of AMY2A. Quantitative real time PCR analysis further confirmed complete loss of expression of the AMY2A gene located at the 1p21.1 region. We demonstrated that AMY2A, a possible TSG, is frequently silenced in GC deletion 1p21.1. The identified gene could provide a basis for further functional validation and may lead to the identification of novel candidates for tumorigenesis and targeted therapies in GC.

Identification of novel candidate target genes, including EPHB3, MASP1 and SST at 3q26.2-q29 in squamous cell carcinoma of the lung.

BACKGROUND: The underlying genetic alterations for squamous cell carcinoma (SCC) and adenocarcinoma (AC) carcinogenesis are largely unknown. METHODS: High-resolution array- CGH was performed to identify the differences in the patterns of genomic imbalances between SCC and AC of non-small cell lung cancer (NSCLC). RESULTS: On a genome-wide profile, SCCs showed higher frequency of gains than ACs (p = 0.067). More specifically, statistically significant differences were observed across the histologic subtypes for gains at 2q14.2, 3q26.2-q29, 12p13.2-p13.33, and 19p13.3, as well as losses at 3p26.2-p26.3, 16p13.11, and 17p11.2 in SCC, and gains at 7q22.1 and losses at 15q22.2-q25.2 occurred in AC (P < 0.05). The most striking difference between SCC and AC was gains at the 3q26.2-q29, occurring in 86% (19/22) of SCCs, but in only 21% (3/14) of ACs. Many significant genes at the 3q26.2-q29 regions previously linked to a specific histology, such as EVI1,MDS1, PIK3CA and TP73L, were observed in SCC (P < 0.05). In addition, we identified the following possible target genes (> 30% of patients) at 3q26.2-q29: LOC389174 (3q26.2),KCNMB3 (3q26.32),EPHB3 (3q27.1), MASP1 and SST (3q27.3), LPP and FGF12 (3q28), and OPA1,KIAA022,LOC220729, LOC440996,LOC440997, and LOC440998 (3q29), all of which were significantly targeted in SCC (P < 0.05). Among these same genes, high-level amplifications were detected for the gene, EPHB3, at 3q27.1, and MASP1 and SST, at 3q27.3 (18, 18, and 14%, respectively). Quantitative real time PCR demonstrated array CGH detected potential candidate genes that were over expressed in SCCs. CONCLUSION: Using whole-genome array CGH, we have successfully identified significant differences and unique information of chromosomal signatures prevalent between the SCC and AC subtypes of NSCLC. The newly identified candidate target genes may prove to be highly attractive candidate molecular markers for the classification of NSCLC histologic subtypes, and could potentially contribute to the pathogenesis of the squamous cell carcinoma of the lung.

Gain of the EGFR gene located on 7p12 is a frequent and early event in squamous cell carcinoma of the lung.

Identification of molecular alterations in biological fluids has been proposed as a powerful tool for cancer diagnosis. The purpose of this study was to identify cells that carry chromosomal alterations indicative of malignancy-specifically, gains in the loci 5p15.2 (D5S23, D5S721), 6p11 approximately q11, 7p12 (EGFR), and 8q24.12 approximately q24.13 (MYC)-for the detection of lung cancer using induced sputum. The overall sensitivity of the multicolor fluorescence in situ hybridization (FISH) assay from 52 lung cancer patients was 71% and the specificity was 100% (15 of 15). The most frequently detected gains were at 7p12 (EGFR) in 17 of 24 completely resectable early-stage (II+IIIA) non-small cell lung cancers (NSCLC) (71%). There was a statistically significant increase in the proportion of cases with gains of EGFR in squamous cell carcinomas (SCC), compared with adenocarcinomas (AC) (82 vs. 43%, respectively; P = 0.017), and a higher average EGFR gene copy number in the SCCs than in the ACs (5.04 vs. 3.78, respectively; P = 0.013) in 41 NSCLCs. Conversely, a gain at the 6p11 approximately q11 and 8q24.12 approximately q24.13 (MYC) regions appears to have a higher frequency of gain in the ACs (71 and 86%, respectively) than in the SCCs (48 and 56%, respectively). The results of this study showed the potential utility of the LAVysion FISH assay for the detection of lung cancer by a noninvasive technique based on the analysis of genetic alterations of induced sputum. Defining abnormalities in sputum specimens as FISH aneusomy may be a possible diagnostic method for the early detection of lung cancer in screening of high-risk populations and monitoring for recurrence.

Gain at chromosomal region 5p15.33, containing TERT, is the most frequent genetic event in early stages of non-small cell lung cancer.

Chromosomal imbalances resulting in altered gene dosage play a role in the molecular pathogenesis of non-small cell lung cancer (NSCLC), but the target genes remain to be identified. To identify early-stage genetic events that drive progression of NSCLC, we conducted a high-resolution array comparative genomic hybridization (CGH) study, using an array of 4,046 bacterial artificial chromosome clones to screen for DNA copy number changes associated with individual genes in 36 tumors obtained from patients in early stages of NSCLC. Multiple early genetic events occurring on chromosome 5p were identified, with a minimal detection region at 5p15.33 approximately 12. The most frequent finding involved gain of 5p15.33, observed in 15 of 19 stage I (A+B) cancers (79%) and in 28 of the total 36 NSCLC cases (78%). This locus harbors the genes TERT, SLC6A19, and SLC6A18 and is a telomeric boundary at bacterial artificial chromosome (BAC) clone 91_J20. Other potential candidate genes evidencing high numbers of genomic copy number changes (> or =40% of patients) included the following genes, encountered in >50% of 19 stage I (A+B) cancers: CEP72 and TPPP (14 of 19; 74%); AHRR, EXOC3 (previously SEC6L1), SLC9A3, LOC442126, ZDHHC11, BRD9, and TRIP13 (13/19; 68%); and CLPTM1L (alias CRR9), SLC6A3 (previously DAT1), and LOC401169 (10/19; 53%). Fluorescence in situ hybridization validated the array CGH findings. The gain of 5p15.33 is thus one of the most consistent alterations in the early stages of lung cancer, and a series of genes in the critical 5p15.33 region may be used as novel biomarkers for the early detection and classification of lung cancer.

High frequency of genetic alterations in non-small cell lung cancer detected by multi-target fluorescence in situ hybridization.

Detection of genetic alterations could provide a tool as an adjuvant for the diagnosis of non-small cell lung cancer (NSCLC) and to define patients at risk for early relapse. In this study, a multi-target fluorescence in situ hybridization (FISH) assay was conducted to investigate the correlation between the alterations of chromosomes, including 5p15.2, 6p11.1-q11, 7p12, and 8q24.12-q24.13 (LaVysion Test), and clinicopathological variables, and to clarify the potential of the multi-target FISH assay in 37 NSCLC. The most notable finding was the higher frequency of a gain in chromosome 5p15.2 in early-stage (I+IIa) lung cancers. The frequency of the gain was 81.3% (16/22) in stage I tumors. The frequencies of gains in 6p11.1-q11 and 8q24.12-q24.13 were 61.5% (8/13) and 84.6% (11/13) in stage IIIa cancers, as compared with lower frequencies in stage I tumors at 25.0% and 31.3%, respectively. There was also a significant difference in the histological type. Our results suggest that a gain in 6p11.1-q11 and 8q24.12-q24.13 plays an important role in tumor progression and is associated with histological differentiation. On the other hand, gene amplification in the 5p region was one of the most consistent alterations in early-stage lung cancer, and thus a series of genes in the critical 5p15.2 region might potentially associated with the development of lung cancer.

Genetic alterations in primary gastric carcinomas correlated with clinicopathological variables by array comparative genomic hybridization.

Genetic alterations have been recognized as an important event in the carcinogenesis of gastric cancer (GC). We conducted high resolution bacterial artificial chromosome array-comparative genomic hybridization, to elucidate in more detail the genomic alterations, and to establish a pattern of DNA copy number changes with distinct clinical variables in GC. Our results showed some correlations between novel amplified or deleted regions and clinical status. Copy-number gains were frequently detected at 1p, 5p, 7q, 8q, 11p, 16p, 20p and 20q, and losses at 1p, 2q, 4q, 5q, 7q, 9p, 14q, and 18q. Losses at 4q23, 9p23, 14q31.1, or 18q21.1 as well as a gain at 20q12 were correlated with tumor-node-metastasis tumor stage. Losses at 9p23 or 14q31.1 were associated with lymph node status. Metastasis was determined to be related to losses at 4q23 or 4q28.2, as well as losses at 4q15.2, 4q21.21, 4q 28.2, or 14q31.1, with differentiation. One of the notable aspects of this study was that the losses at 4q or 14q could be employed in the evaluation of the metastatic status of GC. Our results should provide a potential resource for the molecular cytogenetic events in GC, and should also provide clues in the hunt for genes associated with GC.

Multitarget fluorescence in situ hybridization and melanoma antigen genes analysis in primary bladder carcinoma.

Conventional urine cytology has a poor prognostic performance for detecting bladder cancer, particularly for low-grade tumors. Fluorescence in situ hybridization (FISH) for chromosomes altered in bladder cancer and testing for antigens selectively expressed in tumors are promising alternatives. This study investigated the use of FISH for detecting aneuploidy of chromosomes 3, 7, 17, and 9p21 and reverse transcriptase PCR (RT-PCR) for the expression of melanoma associated antigen (MAGE) genes for the diagnosis of bladder cancer in voided urine specimens. The two techniques were compared with cystoscopic bladder biopsy results in 47 patients with urothelial cancer and 15 patients with benign prostatic hyperplasia. FISH detected cancer in 42 of 47 patients (89.4%). This was significantly higher than the detection rate 30 of 47 patients (64.3%) by MAGE RT-PCR (P < 0.001). The sensitivity of FISH increased with histologic grade and stage of the tumors, correctly identifying 77.8% of pTa and pTis, 94.1% of pT1, and 100% of Pt2-4 tumors. MAGE, however, showed a decreased sensitivity in high grade advanced tumors; it was positive in 66.7% of pTa and pTis, 70.6% of pT1, and 50% of Pt2-4 tumors. Together, the tests correctly identified urothelial cancer in 46 of 47 patients (97.9%). Combined FISH and MAGE RT-PCR testing may offer a promising alternative to conventional urine cytology in screening high-risk populations and in monitoring bladder cancer patients for recurrent tumor.